Method of inhibiting mutant c-kit

ABSTRACT

The present invention includes a method of reducing or inhibiting the kinase activity of C-KIT mutant tyrosine kinase activity in a cell or a subject, and the use of such compound for treating mutant C-KIT driven cell proliferative disorder(s) in a subject related to using a compound of the present invention:or pharmaceutically acceptable salt thereof, wherein the C-KIT mutation is located in at least one of exon 8, exon 9, exon 11, exon 13, exon 14, or exon 17.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/074,800, filed Oct. 20, 2020, which is a continuation-in-part of U.S.patent application Ser. No. 16/283,190, filed Feb. 22, 2019, now U.S.Pat. No. 10,835,525, which is a continuation-in-part of U.S. patentapplication Ser. No. 15/862,011 filed on Jan. 4, 2018, now U.S. Pat. No.10,251,877 issued Apr. 9, 2019, which is a continuation of patentapplication Ser. No. 15/255,912 filed on Sep. 2, 2016, now U.S. Pat. No.9,889,127, which is a continuation of U.S. patent application Ser. No.14/026,886 filed on Sep. 13, 2013, now U.S. Pat. No. 9,480,683 issuedNov. 1, 2016, which claims priority to U.S. Provisional Application Ser.No. 61/705,838, filed Sep. 26, 2012, the entire contents of which areincorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods of reducing or inhibiting thekinase activity of mutated C-KIT in a cell or a subject, and the use ofsuch methods for preventing or treating cell disorders related to C-KIT.

STATEMENT OF FEDERALLY FUNDED RESEARCH

Not applicable.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

Not applicable.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with its ability to inhibit the mutant form of KIT in thetreatment of KIT dependent diseases.

The c-kit gene is located on locus q11-q12 of the human fourthchromosome and encodes the protein KIT (also known as CD 117), which isa cytokine receptor that is expressed on the surface of a number ofdifferent cells. See Rulina et al., Biochemistry (Moscow). ActivatedLeukemic Oncogenes AML1-ETO and c-kit: Role of Development of AcuteMyeloid Leukemia and Current Approaches for Their Inhibition. 2010;75(13): 1650-1666. KIT is a type III receptor tyrosine kinase of themonomeric receptor family and the transmembrane receptor for stem cellfactor. See Tefferi and Pardanani. Leukemia and Lymphoma, March 2010;51(3): 360-362.

KIT is notably expressed by mast cells, hematopoietic progenitor cells,germ cells, melanocytes, and interstitial cells of Cajal in thegastrointestinal tract and is relevant for normal mast cell development,hematopoiesis, gametogenesis, melanogenesis, and regulation of slowgastric waves. See Miettinen et al. KIT (CD117): A Review on Expressionin Normal and Neoplastic Tissues, and Mutations and theirClinicopathologic Correlation. Appl Immunohistochem Mol Morphol. 2005;13: 205-220.

Activating mutations that give rise to ligand-independent activation ofKIT occur in the juxtamembrane and kinase domains of the gene. See Huget al. ETO Interacting Proteins. Oncogene. 2004; 23(24): 4270-4274.Mutations that lead to an activated form of KIT have been shown to playa role in proliferative disease such as mastocytosis, acute myeloidleukemia, gastrointestinal stromal tumors, sinonasal NK/T-cell lymphoma,seminomas, dysgerminomas, melanomas, and thymic carcinomas.

The currently used targeted agent for the treatment of diseasesassociated with both wild-type and mutated KIT is Imatinib mesylate(also known as GLEEVEC or GLIVIC; Novartis, Basel, Switzerland).Imatinib demonstrates activity against certain transmembrane andjuxta-membrane KIT mutants, namely F522C and V560G, respectively, butthis activity is significantly lowered in common kinase domain mutants,including D816V. See Akin et al. A Novel Form of Mastocytosis Associatedwith a Transmembrane C-KIT Mutation and Response to Imatinib. Blood.2004; 103: 3222-3225; Zermati et al. Effect of Tyrosine Kinase InhibitorSTI571 on the Kinase Activity of Wild-type and Various Mutated C-KITReceptors Found in Mast Cell Neoplasms. Oncogene. 2003; 22: 660-664;Akin et al. Effects of Tyrosine Kinase Inhibitor STI571 on Human MastCells Bearing Wild-type or Mutated C-KIT. Exp Hematol. 2003; 31:686-692; Ma et al. The C-KIT Mutation Causing Human Mastocytosis isResistant to STI571 and Other KIT Kinase Inhibitors; Kinases withEnzymatic Site Mutations Show Different Inhibitor Sensitivity Profilesthan Wild-type Kinases and Those with Regulatory-type Mutations. Blood.2002; 99: 1741-1744. Other investigational inhibitors of KIT mutatedkinases in the art include Dasatinib (Bristol-Myers Squibb (BMS), NewYork, N.Y.), Midostaurin (also known as PKC412; Novartis, Basel,Switzerland), and Masatinib (also known as AB1010; AB Science, France).

KIT-dependent diseases include diseases characterized by KIT mutationsD816F, D816H, D816N, D816Y, D816V, K642E, Y823D, Del 550-558, Del557-561, N822K, V654A, N822H, Del 550-558+V654A, Del557-561+V654A,Ins503AY, V560G, 55bNP, Del557-558, Del W559-560, F522C, Del579, R634W,K642E, T801I, C809G, D820Y, N822K, N822H, Y823D, Y823C and T670I.

SUMMARY OF THE INVENTION

In one embodiment, the present invention includes a method of inhibitingor reducing mutant C-KIT tyrosine kinase activity or expression in asubject suffering from a proliferative disease comprised of identifyingthe subject having a proliferative disease positive for constitutivelyactive mutant C-KIT or over expression of C-KIT comprising administeringto the subject having or suspected to have the proliferative disease, atherapeutically effective amount of the compound of Formula I

or a pharmaceutically acceptable salt or solvate thereof, wherein theC-KIT mutant is located in at least one of exon 8, exon 9, exon 11, exon13, exon 14, or exon 17. In one aspect, the C-KIT mutation is at leastone of T417I, Del 418, Del 418-419, T417I, T417V, T417R, T417N, Y418R,Y418A, Y418G, R420W, W420G, L421G, L421F, D496V, Ins A504_Y505, Del557-558, V559A, V559D, K642E, V560G, V654A, T670I, D816E, D816I, D816V,D820E, D820Y, D820V, N822H, N822K, Y823C, Y823D, A829P, V559D/T670I,V559D/V654A, V560G/D816V, and V560G/N822K mutant C-KIT. In one aspect,the compound is provided for the treatment of secondary mutations,wherein secondary mutations is at least one of T670I, V654A, D816V, andN822K. In another aspect, the proliferative disease is selected from atleast one of mastocytosis, acute myeloid leukemia, gastrointestinalstromal tumors, sinonasal NK/T-cell lymphoma, seminomas, dysreminomas,melanomas, and thymic carcinomas. In another aspect, the therapeuticallyeffective amounts are from about 15 to 500, 25 to 450, 50 to 400, 100 to350, 150 to 300, 200 to 250, 15, 25, 50, 75, 100, 150, 200, 250, 300,400, 450, or 500 mg per day. In another aspect, the compound isadministered at least one of continuously, intermittently, systemically,or locally. In another aspect, the mutant C-KIT is defined further asconstitutively active. In another aspect, the compound is administeredorally, intravenously, or intraperitoneally. In another aspect, theCrenolanib is Crenolanib Besylate, Crenolanib Phosphate, CrenolanibLactate, Crenolanib Hydrochloride, Crenolanib Citrate, CrenolanibAcetate, Crenolanib Toluenesulphonate or Crenolanib Succinate. Inanother aspect, the therapeutically effective amount of compound isadministered up to three times or more a day for as long as the subjectis in need of treatment for a proliferative disease. In another aspect,the compound is provided at least one of sequentially or concomitantly,with another pharmaceutical agent in a newly diagnosed proliferativedisease subject, to maintain remission, or a relapsed/refractoryproliferative disease subject. In another aspect, the compound isprovided as a single agent or in combination with another pharmaceuticalagent in a newly diagnosed proliferative disease subject, to maintainremission, or a relapsed/refractory proliferative disease subject. Inanother aspect, the compound is provided as a single agent or incombination with another pharmaceutical agent in a newly diagnosedproliferative disease pediatric subject, to maintain remission, or arelapsed/refractory proliferative disease pediatric subject. In anotheraspect, the method further comprises the step of determining if thesubject is relapsed/refractory to interferon alpha,2-chlorodoxyadenosine or Imatinib mesylate prior to providing thecompound.

In another embodiment, the present invention includes a method fortreating a subject suffering from a proliferative disease comprising:administering to the subject in need of such treatment a therapeuticallyeffective amount of Crenolanib or a salt thereof, wherein the cellproliferative disorder is characterized by mutant or double mutant C-KITreceptor tyrosine kinase activity, and the C-KIT mutated proliferativedisease is selected from at least one of a mastocytosis, acute myeloidleukemia, gastrointestinal stromal tumors, sinonasal NK/T-cell lymphoma,seminomas, dysgerminomas, melanomas, and thymic carcinomas. wherein themutant or double mutant is at least one of: mutations in exons 8, 9, 11,13, 14, or 17, or mutations at T417I, Del 418, Del 418-419, T417I,T417V, T417R, T417N, Y418R, Y418A, Y418G, R420W, W420G, L421G, L421F,D496V, Ins A504_Y505, Del 557-558, V559A, V559D, K642E, V560G, V654A,T670I, D816E, D816I, D816V, D820E, D820Y, D820V, N822H, N822K, Y823C,Y823D, A829P, V559D/T670I, V559D/V654A, V560G/D816V, and V560G/N822Kmutant C-KIT. In one aspect, the compound is provided for the treatmentof secondary mutations from at least one of the following mutationsT670I, V654A, D816V, and N822K. In another aspect, the compound isadministered orally, intravenously, or intraperitoneally. In anotheraspect, the Crenolanib is Crenolanib Besylate, Crenolanib Phosphate,Crenolanib Lactate, Crenolanib Hydrochloride, Crenolanib Citrate,Crenolanib Acetate, Crenolanib Toluenesulphonate or CrenolanibSuccinate. In another aspect, the therapeutically effective amount ofthe compound is administered up to three times or more a day for as longas the subject is in need of treatment for proliferative disease. Inanother aspect, the Crenolanib is provided at least one of sequentiallyor concomitantly, with another pharmaceutical agent in a newly diagnosedproliferative disease, to maintain remission, or a relapsed/refractoryproliferative disease. In another aspect, the Crenolanib is provided asa single agent or in combination with another pharmaceutical agent fortreatment of a pediatric subject with the proliferative disease. Inanother aspect, the Crenolanib is provided as a single agent to at leastone of post chemotherapeutic or targeted therapy in newly diagnosedproliferative disease. In another aspect, the Crenolanib is provided asa single agent in treatment of subjects with the proliferative diseasethat is either refractory to or has relapsed after treatment withanother chemotherapeutic or targeted therapy. In another aspect, thesubject is refractory to at least one of interferon alpha, 2chlorodoxyadenosine or Imatinib mesylate. In another aspect, the methodfurther comprises the step of determining identifying a subject in needof cancer therapy caused by uncontrolled C-KIT tyrosine kinase activityor expression.

In one embodiment, the present invention includes a method of inhibitingor reducing mutant C-KIT tyrosine kinase activity or expression in asubject suffering from a proliferative disease which comprisesadministering to the subject having or suspected to have theproliferative disease, a therapeutically effective amount of thecompound of Formula I:

or a pharmaceutically acceptable salt or solvate thereof wherein theC-KIT mutant is located in at least one of exon 8, exon 9, exon 11, exon13, exon 14, or exon 17. In one aspect, the C-KIT mutation is at leastone of T417I, Del 418, Del 418-419, T417I, T417V, T417R, T417N, Y418R,Y418A, Y418G, R420W, W420G, L421G, L421F, D496V, Ins A504_Y505, Del557-558, V559A, V559D, K642E, V560G, V654A, T670I, D816E, D816I, D816V,D820E, D820Y, D820V, N822H, N822K, Y823C, Y823D, A829P, V559D/T670I,V559D/V654A, V560G/D816V, and V560G/N822K mutant C-KIT. In one aspect,the compound is provided for the treatment of secondary mutations,wherein the secondary mutations is at least one of T670I, V654A, D816V,and N822K. In another aspect, the proliferative disease is selected fromat least one of mastocytosis, acute myeloid leukemia, gastrointestinalstromal tumors, sinonasal NK/T-cell lymphoma, seminomas, dysreminomas,melanomas, and thymic carcinomas. In another aspect, the therapeuticallyeffective amounts are from about 15 to 500, 25 to 450, 50 to 400, 100 to350, 150 to 300, 200 to 250, 15, 25, 50, 75, 100, 150, 200, 250, 300,400, 450, or 500 mg per day. In another aspect, the compound isadministered at least one of continuously, intermittently, systemically,or locally. In another aspect, the mutated C-KIT is defined further asconstitutively active. In another aspect, the compound is administeredorally, intravenously, or intraperitoneally. In another aspect, theCrenolanib is Crenolanib Besylate, Crenolanib Phosphate, CrenolanibLactate, Crenolanib Hydrochloride, Crenolanib Citrate, CrenolanibAcetate, Crenolanib Toluenesulphonate and Crenolanib Succinate. Inanother aspect, the therapeutically effective amount of the compound isadministered up to three times or more for as long as the subject is inneed of treatment for a proliferative disease. In another aspect, thecomposition is provided at least one of sequentially or concomitantly,with another pharmaceutical agent in a newly diagnosed proliferativedisease subject, or a relapsed/refractory proliferative disease subject.In another aspect, the compound is provided as a single agent or incombination with another pharmaceutical agent in a newly diagnosedproliferative disease subject, to maintain remission, or arelapsed/refractory proliferative disease subject. In another aspect,the compound is provided as a single agent or in combination withanother pharmaceutical agent in a newly diagnosed proliferative diseasepediatric subject, or a relapsed/refractory proliferative diseasepediatric subject. In another aspect, the subject is relapsed/refractoryto Interferon alpha, 2-chlorodoxyadenosine, or Imatinib Mesylate priorto providing the compound.

In another embodiment, the present invention includes a method fortreating a subject suffering from a proliferative disease comprising:administering to the subject in need of such treatment a therapeuticallyeffective amount of Crenolanib or a salt thereof, wherein the cellproliferative disorder is characterized by mutant or double mutant C-KITreceptor tyrosine kinase activity, proliferative disease is selectedfrom at least one of a mastocytosis, acute myeloid leukemia,gastrointestinal stromal tumors, sinonasal NK/T-cell lymphoma,seminomas, dysgerminomas, melanomas, and thymic carcinomas. wherein themutant or double mutant is at least one of: exon 8, exon 9, exon 11,exon 13, exon 14, or exon 17, or T417I, Del 418, Del 418-419, T417I,T417V, T417R, T417N, Y418R, Y418A, Y418G, R420W, W420G, L421G, L421F,D496V, Ins A504_Y505, Del 557-558, V559A, V559D, K642E, V560G, V654A,T670I, D816E, D816I, D816V, D820E, D820Y, D820V, N822H, N822K, Y823C,Y823D, A829P, V559D/T670I, V559D/V654A, V560G/D816V, and V560G/N822Kmutant C-KIT. In one aspect, the compound is provided for the treatmentof secondary mutations from at least one of the following mutationsT670I, V654A, D816V, and N822K. In another aspect, the compound isadministered orally, intravenously, or intraperitoneally. In anotheraspect, the Crenolanib is Crenolanib Besylate, Crenolanib Phosphate,Crenolanib Lactate, Crenolanib Hydrochloride, Crenolanib Citrate,Crenolanib Acetate, Crenolanib Toluenesulphonate or CrenolanibSuccinate. In another aspect, the therapeutically effective amount ofthe compound is administered up to three times or more a day for as longas the subject is in need of treatment for proliferative disease. Inanother aspect, the Crenolanib is provided at least one of sequentiallyor concomitantly, with another pharmaceutical agent in a newly diagnosedproliferative disease, to maintain remission, or a relapsed/refractoryproliferative disease. In another aspect, the Crenolanib is provided asa single agent or in combination with another pharmaceutical agent fortreatment of a pediatric subject with the proliferative disease. Inanother aspect, the Crenolanib is provided as a single agent to at leastone of post chemotherapeutic or targeted therapy in newly diagnosedproliferative disease. In another aspect, the Crenolanib is provided asa single agent in treatment of subjects with the proliferative diseasethat is either refractory to or has relapsed after treatment withanother chemotherapeutic or targeted therapy. In another aspect, thesubject is refractory to at least one of interferon alpha, 2chlorodoxyadenosine or Imatinib mesylate. In another aspect, the methodfurther comprises the step of determining identifying a subject in needof cancer therapy caused by uncontrolled C-KIT tyrosine kinase activityor expression.

The present invention relates to the inhibition of domain mutated KITand correlates with the treatment of such diseases driven by mutatedKIT. The present invention includes a method of inhibiting or reducingmutated C-KIT tyrosine kinase activity or expression in a subjectsuffering from a proliferative disease driven by mutant C-KIT whichcomprises administering to the subject having a proliferative disease, atherapeutically effective amount of the compound of Formula I:

or a pharmaceutically acceptable salt or solvate thereof. In one aspect,the therapeutically effective amounts of the present invention are fromabout 15 to 500 mg per day. In another aspect, the compound isadministered at least one of continuously, intermittently, systemically,or locally. In another aspect, the mutated C-KIT is defined further as amutated C-KIT that is constitutively active. In another aspect, thecompound is administered orally, intravenously, or intraperitoneally. Inanother aspect, the Crenolanib is Crenolanib Besylate, CrenolanibPhosphate, Crenolanib Lactate, Crenolanib Hydrochloride, CrenolanibCitrate, Crenolanib Acetate, Crenolanib Toluenesulphonate and CrenolanibSuccinate. In another aspect, the C-KIT mutation is at least one of exon8, exon 9, exon 11, exon 13, exon 14, or exon 17. In one aspect, theC-KIT mutation is at least one of T417I, Del 418, Del 418-419, T417I,T417V, T417R, T417N, Y418R, Y418A, Y418G, R420W, W420G, L421G, L421F,D496V, Ins A504_Y505, D816F, D816H, D816N, D816Y, D816V, K642E, Y823D,Del 550-558, Del 557-561, N822K, V654A, N822H, Del 550-558+V654A,Del557-561+V654A, Ins503AY, V560G, 55bNP, Del557-558, Del W559-560,F522C, Del579, R634W, K642E, T801I, C809G, D820Y, N822K, N822H, Y823D,Y823C and T670I. In another aspect, the therapeutically effective amountof the compound is administered up to three times or more for as long asthe subject is in need of treatment for the C-KIT mutant activatedproliferative disease. In another aspect, the composition is provided atleast one of sequentially or concomitantly, with another pharmaceuticalagent in a newly diagnosed proliferative disease subject, or arelapsed/refractory proliferative disease subject. In another aspect,the compound is provided as a single agent or in combination withanother pharmaceutical agent in a newly diagnosed proliferative diseasesubject, or a relapsed/refractory proliferative disease subject. Inanother aspect, the compound is provided as a single agent or incombination with another pharmaceutical agent in a newly diagnosedproliferative disease pediatric subject, or a relapsed/refractoryproliferative disease pediatric subject. In another aspect, the subjectis relapsed/refractory to Interferon alpha, 2-chlorodoxyadenosine, orImatinib Mesylate.

In another embodiment, the present invention includes a method fortreating a subject suffering from a C-KIT mutant driven proliferativedisease comprising: administering to the subject in need of suchtreatment a therapeutically effective amount of the present invention ora salt thereof, wherein the cell proliferative disorder is characterizedby C-KIT mutant receptor tyrosine kinase activity, the proliferativedisease is selected from at least one of mastocytosis, acute myeloidleukemia, gastrointestinal stromal tumors, sinonasal NK/T-cell lymphoma,seminomas, dysgerminomas, melanomas, and thymic carcinomas. In anotheraspect, the Crenolanib is Crenolanib Besylate, Crenolanib Phosphate,Crenolanib Lactate, Crenolanib Hydrochloride, Crenolanib Citrate,Crenolanib Acetate, Crenolanib Toluenesulphonate and CrenolanibSuccinate. In another aspect, the C-KIT mutation is one of located in atleast one of exon 8, exon 9, exon 11, exon 13, exon 14, or exon 17,T417I, Del 418, Del 418-419, T417I, T417V, T417R, T417N, Y418R, Y418A,Y418G, R420W, W420G, L421G, L421F, D496V, Ins A504_Y505, D816F, D816H,D816N, D816Y, D816V, K642E, Y823D, Del 550-558, Del 557-561, N822K,V654A, N822H, Del 550-558+V654A, Del557-561+V654A, Ins503AY, V560G,55bNP, Del557-558, Del W559-560, F522C, Del579, R634W, K642E, T801I,C809G, D820Y, N822K, N822H, Y823D, Y823C and T670I. In another aspect,Crenolanib is provided at least one of sequentially or concomitantly,with another pharmaceutical agent in a newly diagnosed proliferativedisease, or a relapsed/refractory proliferative disease. In anotheraspect, Crenolanib is provided as a single agent or in combination withanother pharmaceutical agent for treatment of a pediatric subject withthe proliferative disease. In another aspect, Crenolanib is provided asa single agent either concomitantly or sequential with achemotherapeutic or targeted therapy, in newly diagnosed proliferativedisease. In another aspect, Crenolanib is provided as a single agent intreatment of a subject with the proliferative disease that is eitherrefractory to, or has relapsed after, chemotherapeutic or targetedtherapy. In another aspect, the subject is refractory to at least one ofinterferon alpha, 2-chlorodoxyadenosine or Imatinib Mesylate.

The present invention provides methods of reducing or inhibiting thekinase activity of mutant C-KIT in a cell or a subject, and the use ofsuch methods treating cell proliferative disorder (s) driven by mutantC-KIT. Other features and advantages of the invention will be apparentfrom the following detailed description of the invention and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A and 1B show the replicates of standard dose-response curves forKd determination of the besylate salt of the present invention for FLT3D816H (left and right panels are separate replicates). The amount ofkinase measured by qPCR (signal; y-axis) is plotted against thecorresponding crenolanib concentration in nanomolar in log 10 scale(x-axis);

FIGS. 2A and 2B show the replicates of standard dose-response curves forKd determination of the besylate salt of the present invention for FLT3D816V (left and right panels are separate replicates). The amount ofkinase measured by qPCR (signal; y-axis) is plotted against thecorresponding crenolanib concentration in nanomolar in log 10 scale(x-axis);

FIG. 3 shows the binding constants of the besylate salt of the presentinvention compared to other KIT tyrosine kinase inhibitors for theconstitutively active KIT D816H mutation;

FIG. 4 shows the binding constants of the besylate salt of the presentinvention compared to other KIT tyrosine kinase inhibitors for theconstitutively active FLT3 D816H mutation; and

FIG. 5 shows the dose-response curve (n=2) for IC50 determination of thebesylate salt of the present invention for FLT3 D835Y. The activity ofthe besylate salt of crenolanib is plotted against the correspondingmolar concentration in log 10 scale.

FIG. 6 is a graph that shows the percent inhibition of cKIT(V559A)Enzymatic activity by crenolanib.

FIG. 7 is a graph that shows the percent inhibition of cKIT(V559D)Enzymatic activity by crenolanib.

FIG. 8 is a graph that shows the percent inhibition of cKIT(V654A)Enzymatic activity by crenolanib.

FIG. 9 is a graph that shows the percent inhibition of cKIT(D816E)Enzymatic activity by crenolanib.

FIG. 10 is a graph that shows the percent inhibition of cKIT(D816I)Enzymatic activity by crenolanib.

FIG. 11 is a graph that shows the percent inhibition of cKIT(D816V)Enzymatic activity by crenolanib.

FIG. 12 is a graph that shows the percent inhibition of cKIT(D820E)Enzymatic activity by crenolanib.

FIG. 13 is a graph that shows the percent inhibition of cKIT(D820V)Enzymatic activity by crenolanib.

FIG. 14 is a graph that shows the percent inhibition of cKIT(A829P)Enzymatic activity by crenolanib.

FIG. 15 is a graph that shows the percent inhibition ofcKIT(V559D/Y670I) Enzymatic activity by crenolanib.

FIG. 16 is a graph that shows the percent inhibition ofcKIT(V559D/V654A) Enzymatic activity by crenolanib.

FIG. 17 is a graph that shows the percent inhibition ofcKIT(V560G/D816V) Enzymatic activity by crenolanib.

FIG. 18 is a graph that shows the percent inhibition ofcKIT(V560G/N882K) Enzymatic activity by crenolanib.

FIG. 19 is a map of the locations of the mutations in relation to thekinase domains of c-kit.

FIG. 20 is a map of the locations of the mutations in relation to thekinase domains of c-kit.

FIGS. 21A and 21B show (FIG. 21A) drug sensitivity tested against BaF3cell lines expressing mutant c-kit D816V and N882K, with 21B showing theIC50 values for crenolanib shown in FIG. 21A.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The present invention comprises the use of the compounds of the presentinvention to inhibit mutant C-KIT kinase activity in a cell or asubject, or to treat disorders related to mutant C-KIT kinase activityor expression in a subject.

In one embodiment to this aspect, the present invention provides amethod for reducing or inhibiting the kinase activity of mutant C-KIT ina cell comprising the step of contacting the cell with a compound of thepresent invention. The present invention also provides a method forreducing or inhibiting the kinase activity of mutant C-KIT in a subjectcomprising the step of administering a compound of the present inventionto the subject. The present invention further provides a method ofinhibiting cell proliferation in a cell comprising the step ofcontacting the cell with a compound of the present invention.

As used herein, the term “subject” refers to an animal, such as a mammalor a human, who has been the object of treatment, observation orexperiment.

As used herein, the term “contacting” refers to the addition of thepresent invention or pharmaceutically acceptable salt to cells such thatthe compound is taken up by the cell.

In other embodiments to this aspect, the present invention providestherapeutic methods for treating a subject with a cell proliferativedisorder driven by aberrant kinase activity of mutant C-KIT.

As used herein, the term “therapeutically effective amount” as usedherein, refers to an amount of active compound or pharmaceutical saltthat elicits the biological or medicinal response in a subject that isbeing sought by a researcher, veterinarian, medical doctor or otherclinician, which includes alleviation of the symptoms of the disease ordisorder being treated.

Methods for determining therapeutically effective doses forpharmaceutical compositions comprising a compound of the presentinvention are known in the art.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombinations of the specified ingredients in the specified amounts.

As used herein, the terms “disorder related to mutant C-KIT,” or“disorders related to C-KIT mutated receptor tyrosine kinase,” or“mutant C-KIT driven cell proliferative disorder” includes diseasesassociated with or implicating mutant C-KIT activity, for example,mutations leading to constitutive activation of C-KIT. Examples of“disorders related to mutant C-KIT” include disorders resulting fromover stimulation of FLT3 due to mutations in C-KIT.

Mutations in KIT occur in certain hotspot regions of the gene. Thesehotspots include exons 8, 9, 11, 13, 14, and 17. See Miettinen et al.KIT (CD117): A Review on Expression in Normal and Neoplastic Tissues,and Mutations and their Clinicopathologic Correlation. ApplImmunohistochem Mol Morphol. 2005; 13: 205-220. See Rulina et al.,Biochemistry (Moscow). Activated Leukemic Oncogenes AML1-ETO and c-kit:Role of Development of Acute Myeloid Leukemia and Current Approaches forTheir Inhibition. 2010; 75(13): 1650-1666, relevant portionsincorporated herein by reference. Mutations in these exons areactivating, and lead to ligand independent signaling. Mutationsinvolving amino acid resides in these exons have been identified inproliferative diseases including gastrointestinal stromal tumors, acutemyeloid leukemia, systemic mastocytosis, thymic carcinomas, seminomasand others, with varying frequencies. Identified mutations include:T417I, Del 418, Del 418-419, T417I, T417V, T417R, T417N, Y418R, Y418A,Y418G, R420W, W420G, L421G, and L421F in exon 8; D496V, Ins A504_Y505,and F522C in exon 9; Del 550-558, Del 557-561, Del557-558, Del W559-560,V560G, and Del579 in exon 11; V654A, V654A, R634W, K642E, in exon 13;T670I in exon 14; T801I, C809G, D816F, D816H, D816N, D816Y, D816V,D820Y, N822K, N822H, Y823D, and Y823C in exon 17. Compound mutationsincluding Del 550-558+V654A and Del557-561+V654A have also beenreported.

The term “cell proliferative disorders” refers to excess cellproliferation of one or more subset of cells in a multicellular organismresulting in harm (i.e. discomfort or decreased life expectancy) to themulticellular organism. Cell proliferative disorders can occur indifferent types of animals and humans. Examples of cell proliferativedisorders are mastocytosis, acute myeloid leukemia, gastrointestinalstromal tumors, sinonasal NK/T-cell lymphoma, seminomas, dysgerminomas,melanomas, and thymic carcinomas.

In a further embodiment, the present invention can be combined withanother therapy as a combination therapy for treating the onset of acell proliferative disorder related to mutant C-KIT in a subject. Thecombination therapy comprises the administration of a therapeuticallyeffective amount of a compound of the present invention and one or moreother anti-cell proliferation therapies including, but not limited to,chemotherapy and targeted therapy.

In an embodiment of the present invention, a compound of the presentinvention may be administered in combination with chemotherapy. Usedherein, chemotherapy refers to a therapy involving a chemotherapeuticagent. A variety of chemotherapeutic agents may be used in combinationwith the present invention. By way of example only, taxane compounds,specifically docetaxel, is safely administered in combination with acompound of the present invention in a dosage of 75 mg per square meter(mg/m²) of body surface area.

Chemotherapy is known to those skilled in the art. The appropriatedosage and scheme for chemotherapy will be similar to those alreadyemployed in clinical therapies wherein the chemotherapy is delivered incombination with other therapies or used alone.

In another embodiment of the present invention, compounds of the presentinvention may be administered in combination with radiation therapy. Asused herein, the term “radiation therapy” refers to a therapy thatcomprises the exposure of a subject in need to radiation. Radiationtherapy is known to those skilled in the art. The appropriate dosage andscheme for radiation therapy will be similar to those already employedin clinical therapies wherein the radiation therapy is delivered incombination with other therapies or used alone.

In another embodiment of the present invention, the compounds of thepresent invention may be administered in combination with a targetedtherapy. As used herein, the term “targeted therapy” refers to a therapytargeting a particular class of proteins involved in tumor developmentor oncogenic signaling. For example, tyrosine kinase inhibitors againstvascular endothelial growth factor have been used in treating cancers.

The present invention also includes methods that include the use of asecond pharmaceutical agent in addition to compounds of the presentinvention, the two may be administered simultaneously or sequentially(in either order).

In one embodiment, the present invention therapeutically effectiveamounts of the compound having formula I:

or a pharmaceutically acceptable salt or solvate thereof, in atherapeutically effective amount against a proliferative disease isselected from at least one of mastocytosis, acute myeloid leukemia,gastrointestinal stromal tumors, sinonasal NK/T-cell lymphoma,seminomas, dysgerminomas, melanomas, and thymic carcinomas.Pharmaceutically acceptable salts such as hydrochloride, phosphate andlactate are prepared in a manner similar to the benzenesulfonate saltand are well known to those of moderate skill in the art.

Compounds of the present invention may be administered to a subjectsystemically, for example, orally, intravenously, subcutaneously,intramuscular, intradermal or parenterally. The compounds of the presentinvention can also be administered to a subject locally.

Compounds of the present invention may be formulated for slow-release orfast-release with the objective of maintaining contact of compounds ofthe present invention with targeted tissues for a desired range of time.

Compositions suitable for oral administration include solid forms, suchas pills, tablets, caplets, capsules, granules, and powders, liquidforms, such as solutions, emulsions, and suspensions. Forms useful forparenteral administration include sterile solutions, emulsions andsuspensions.

The daily dosage of the compounds of the present invention may be variedover a wide range from 15 to 500, 25 to 450, 50 to 400, 100 to 350, 150to 300, 200 to 250, 15, 25, 50, 75, 100, 150, 200, 250, 300, 400, 450,or 500 mg per day. The compounds of the present invention may beadministered on a daily regimen, once, twice, three or more times perday. Optimal doses to be administered may be determined by those skilledin the art, and will vary with the compound of the present inventionused, the mode of administration, the time of administration, thestrength of the preparation, and the details of the disease condition.One or more factors associated with subject characteristics, such asage, weight, and diet will call for dosage adjustments. Techniques andcompositions for making useful dosage forms using the Crenolanib aredescribed in one or more of the following references: Anderson, PhilipO.; Knoben, James E.; Troutman, William G, eds., Handbook of ClinicalDrug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds.,Principles of Drug Action, Third Edition, Churchill Livingston, N.Y.,1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition,McGraw Hill, 20037ybg; Goodman and Gilman, eds., The PharmacologicalBasis of Therapeutics, Tenth Edition, McGraw Hill, 2001; RemingtonsPharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins, 2000;Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (ThePharmaceutical Press, London, 1999); relevant portions incorporatedherein by reference.

A dosage unit for use of Crenolanib, may be a single compound ormixtures thereof with other compounds, e.g., a potentiator. Thecompounds may be mixed together, form ionic or even covalent bonds. Thecompounds of the present invention may be administered in oral,intravenous (bolus or infusion), intraperitoneal, subcutaneous, orintramuscular form, all using dosage forms well known to those ofordinary skill in the pharmaceutical arts. Depending on the particularlocation or method of delivery, different dosage forms, e.g., tablets,capsules, pills, powders, granules, elixirs, tinctures, suspensions,syrups, and emulsions may be used to provide the compounds of thepresent invention to a patient in need of therapy that includes thecompound of Formula I.

The Crenolanib is typically administered in admixture with suitablepharmaceutical salts, buffers, diluents, extenders, excipients and/orcarriers (collectively referred to herein as a pharmaceuticallyacceptable carrier or carrier materials) selected based on the intendedform of administration and as consistent with conventionalpharmaceutical practices. Depending on the best location foradministration, the Crenolanib may be formulated to provide, e.g.,maximum and/or consistent dosing for the particular form for oral,rectal, topical, intravenous injection or parenteral administration.While the Crenolanib may be administered alone, it will generally beprovided in a stable salt form mixed with a pharmaceutically acceptablecarrier. The carrier may be solid or liquid, depending on the typeand/or location of administration selected.

Preparation of the compounds of the present invention. General syntheticmethods which may be referred to for preparing the compounds of formulaI are provided in U.S. Pat. No. 5,990,146 (issued Nov. 23, 1999)(Warner-Lambert Co.) and PCT published application numbers WO 99/16755(published Apr. 8, 1999) (Merck & Co.) WO 01/40217 (published Jul. 7,2001) (Pfizer, Inc.), US Patent Application Publication No. US2005/0124599 (Pfizer, Inc.) and U.S. Pat. No. 7,183,414 (Pfizer, Inc.),relevant portions incorporated herein by reference.

Pharmaceutically acceptable salts such as hydrochloride, phosphate andlactate are prepared in a manner similar to the benzenesulfonate saltand are well known to those of moderate skill in the art. The followingrepresentative compounds of the present invention are for exemplarypurposes only and is in no way meant to limit the invention.

Biological Activity.

In Vitro Assays. The following representative in vitro assays wereperformed in determining the C-KIT biological activity of the presentinvention. These are given to illustrate the invention in a non-limitingfashion.

Inhibition of mutant C-KIT enzyme activity exemplifies the specificinhibition of the mutant C-KIT enzyme and cellular processes that aredependent on mutant C-KIT activity. All of the examples herein showsignificant and specific inhibition of mutant C-KIT kinase andC-KIT-dependent cellular responses.

Competitive binding assay. To determine the activity of the presentinvention in an in vitro kinase assay. Inhibition of the kinase domainof the mutant human C-KIT receptor was performed using the KINOMEscanKdelect assay protocol. The KINOMEscan platform utilizes ahigh-throughput competitive binding technology. The assay was performedby combining DNA-tagged kinase, immobilized ligand, and the presentinvention. The ability of the present invention to compete withimmobilized ligand was measured using quantitative PCR of the DNA tag.The competition binding assay was used to evaluate the present inventionagainst a panel of 96 human protein kinases.

Kinase-tagged T7 phage strains were grown in parallel in 24-well blocksin an E. coli host derived from the BL21 strain. E. coli were grown tolog phase and infected with T7 phage from a frozen stock and incubatedwith shaking at 32 degrees Celsius until lysis. The lysates were thencentrifuged and filtered. The remaining kinases were produced in HEK-293cells and tagged with DNA for quantitative PCR detection. Affinityresins for the kinase assay were generated by treatingstreptavidin-coated magnetic beads with biotinylated small moleculeligands for 30 minutes at room temperature. The liganded beads wereblocked with excess biotin and washed with blocking buffer consisting ofSea Block, 1% Bovine Serum Albumin (BSA) 0.05% Tween 20, 1 mMDithithreitol (DTT) in order to reduce non-specific phage binding. An11-point 3-fold serial dilution of the present invention was prepared asa 40× stock in 100% Dimethyl sulfoxide (DMSO) and diluted to 1× directlyinto the assay.

Binding reactions were initiated by combining the liganded affinitybeads, kinases, and the present invention in 1× binding bufferconsisting of 20% Sea Block, 0.17 Phosphate Buffered Saline (PBS), 0.05%Tween 20, 6 mM DTT. All reactions were performed in polypropylene384-well plates in a final volume of 0.04 mL. The plates were incubatedfor 1 hour while shaking at room temperature. The affinity beads werewashed with 1×PBS and 0.05% Tween 20 buffer, then re-suspended inelution buffer consisting of 1×PBS, 0.05% Tween 20, 0.5 uMnon-biotinylated affinity ligand. Following re-suspension, the affinitybeads were incubated at room temperature with shaking. The elutantkinase concentration was then measured by quantitative PCR.

Binding constants (Kds) were calculated with a standard dose-responsecurve using the Hill equation. Curves were fitted using a non-linearleast square fit with the Levenberg-Marquardt algorithm. Kds of thepresent invention were compared to both a negative DMSO control and apositive control compound. The binding affinity of the present inventionwas visualized using the compound profile visualization interaction map,TREEspot.

Direct enzyme phosphorylation assay. The Millipore Kinase IC50 Profilerassay was used to screen the present invention against a panel of normalC-KIT and mutated C-KIT kinases. For assays of both kinases, the C-KITenzyme was incubated with 8 mM of 3-(N-morpholino)propanesulfonic acid(MOPS) at a pH of 7.0, 0.2 mM Ethylenediaminetetraacetic acid (EDTA), 50uM, a synthetic Abl peptide substrate EAIYAAPFAKKK, 10 mM MgAcetate and[7-33P-ATP]. The reaction was initiated by the addition of MgATp mix.The reaction mixture was incubated for 40 minutes at room temperatureand halted by the addition of 3% phosphoric acid solution. 10 uL of thereaction solution was spotted on P30 filtermat and washed three times in75 mM phosphoric acid for 5 minutes and then once in methanol prior todrying and scintillation counting. The scintillation values for eachreplicate, including positive and negative controls, were analyzed usingXLFit version 5.1 to determine the IC50 values for the present inventionagainst normal and mutated C-KIT.

Biological data for the C-KIT-D816 mutation. The activity dose-responsecurves of the besylate salt of the present invention against C-KITtyrosine kinase domain mutations D816H and D816V are presented inreplicate in FIGS. 1 and 2 (left and right panels are separatereplicates), respectively. In both FIGS. 3 and 4 , the activity of thepresent invention for the C-KIT D816H and C-KIT D816V mutations iscompared against other inhibitors known in the art. All bindingconstants are presented in nanomolar concentration. See Davis M I, HuntJ P, Herrgard S, et al. Comprehensive analysis of kinase inhibitorselectivity. Nat Biotechnol 2011; 29:1046-51. The binding constant (Kd)of the besylate salt of the present invention for the C-KIT D816Hmutation is 5.4 nM and 2.5 nM for the C-KIT D816V mutation. Whencomparing the Kd of the besylate salt of the present invention for theC-KIT D816H and D816V mutations and other inhibitors in the art, thebesylate form of the present invention had a range between one and onehundred three times greater affinity for the C-KIT D816H mutation arange between one and three hundred ninety-two times greater for theC-KIT D816V mutation. Against Imatinib mesylate, the besylate form ofthe present invention had more than one hundred three times greateraffinity for the C-KIT D816H mutation and three hundred ninety-two timesmore affinity for the C-KIT DB816V mutation than Imatinib Mesylate(D816H Kd=560 nM and D816V Kd=980 nM).

The activity of the besylate salt of the present invention wasdetermined using a direct enzymatic Millipore IC50 profiler assay. Inthe direct enzymatic measurement assay, the IC50 of the besylate salt ofthe current invention against the C-KIT D816H mutation was 7 nM (FIG. 5).

FIG. 6 is a graph that shows the percent inhibition of cKIT(V599A)enzymatic activity by crenolanib.

FIG. 7 is a graph that shows the percent inhibition of cKIT(V559D)Enzymatic activity by crenolanib. FIG. 8 is a graph that shows thepercent inhibition of cKIT(V654A) Enzymatic activity by crenolanib. FIG.9 is a graph that shows the percent inhibition of cKIT(D816E) Enzymaticactivity by crenolanib. FIG. 10 is a graph that shows the percentinhibition of cKIT(D816I) Enzymatic activity by crenolanib. FIG. 11 is agraph that shows the percent inhibition of cKIT(D816V) Enzymaticactivity by crenolanib. FIG. 12 is a graph that shows the percentinhibition of cKIT(D820E) Enzymatic activity by crenolanib. FIG. 13 is agraph that shows the percent inhibition of cKIT(D820V) Enzymaticactivity by crenolanib. FIG. 14 is a graph that shows the percentinhibition of cKIT(A829P) Enzymatic activity by crenolanib. FIG. 15 is agraph that shows the percent inhibition of cKIT(V559D/Y670I) Enzymaticactivity by crenolanib. FIG. 16 is a graph that shows the percentinhibition of cKIT(V559D/V654A) Enzymatic activity by crenolanib. FIG.17 is a graph that shows the percent inhibition of cKIT(V560G/D816V)Enzymatic activity by crenolanib. FIG. 18 is a graph that shows thepercent inhibition of cKIT(V560G/N882K) Enzymatic activity bycrenolanib. FIG. 19 is a map of the locations of the mutations inrelation to the kinase domains of c-kit. FIG. 20 is a map of thelocations of the mutations in relation to the kinase domains of c-kit.FIGS. 21A and 21 b show (FIG. 21A) drug sensitivity tested against BaF3cell lines expressing mutant c-kit D816V and N882K, with 21B showing theIC50 values for crenolanib shown in FIG. 21A.

Table 1 shows the results with crenolanib besylate in two differentassays of a kinase assay measuring activity as measured by % enzymeactivity.

% Enzyme Activity (relative to DMSO controls) IC50 (M) Crenolanibbesylate API Control Kinases Data 1 Data 2 Average Cmpd Control Cmpd IDc-Kit 93.80 93.61 93.71 1.02E−07 Stauro sporine c-Kit (A829P) 20.9

20.39 20.68 2.48E−09 Stauro sporine c-Kit (d557-558) 5.93 5.61 5.772.45E−10 Stauro sporine c-Kit (D816E) 6.

5 6.54 6.60 1.36E−10 Stauro sporine c-Kit (D816F ) 11.04 9.74 10.392.11E−10 Stauro sporine c-Kit (D818

) 2.57 2.36 2.47 3.47E−10 Stauro sporine c-Kit (D816

) 5.27 5.16 5.22 6.48E−10 Stauro sporine c-Kit (D816V) 1.47 1.10 1.291.63E−10 Stauro sporine c-Kit (D816Y) 3.91 3.39 3.65 1.93E−10 Staurosporine c-Kit (D820E) 9.

7 9.37 9.37 8.17E−10 Stauro sporine c-Kit (D820Y) 4.86 3.39 4.132.44E−10 Stauro sporine c-Kit (K642E) 36.20 35.91 36.06 3.13E−09 Staurosporine c-KI (T670

) 65.23 61.78 63.50 7.43E−09 Stauro sporine c-KIL (V559A) 6.88 6.67 6.771.39E−10 Stauro sporine c-Kit (V559D) 8.97 8.82 8.89 1.13E−09 Staurosporine c-Kit (V559D/T670

) 17.67 17.24 17.46 2.48E−10 Stauro sporine c-Kit (V559D/V654A) 25.9725.27 25.62 1.13E−09 Stauro sporine c-Kit (V560G ) 6.97 6.60 6.784.43E−10 Stauro sporine c-Kit (V560G/D816V) 2.99 2.49 2.74 1.47E−10Stauro sporine c Kit (V560G/N822K) 5.71 5.19 5.45 1.77E−10 Staurosporine c-Kit (V654A) 91.94 90.29 91.12 1.16E−08 Stauro sporine c-Kit(Y8230) 13.73 13.35 13.54 8.64E−10 Stauro sporine FMS 10.94 10.04 10.491.27E−09 Stauro sporine PDGF Ra 13.73 13.37 13.55 4.06E−09 Staurosporine PDGF Ra (D842V) 0.20 −3.56 −1.68 7.32E−10 Stauro sporine PDGF Ra(T674

) 10.59 10.54 10.57 2.33E−09 Stauro sporine PDGF Ra (V561D) 9.75 9.589.67 5.04E−09 Stauro sporine PDGF Ra-FIP1L1 9.62 9.10 9.36 2.78E−09Stauro sporine PDGF Rb 11.13 11.09 11.11 2.72E−09 Stauro sporine PDGFRb-TPM3 3.05 2.41 2.73 1.19E−09 Stauro sporine

indicates data missing or illegible when filed

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skilled in the art recognizethe modified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

-   Rulina et al., Biochemistry (Moscow). Activated Leukemic Oncogenes    AML1-ETO and c-kit: Role of Development of Acute Myeloid Leukemia    and Current Approaches for Their Inhibition. 2010; 75(13):    1650-1666.-   Tefferi and Pardanani. Targeted Therapy in KIT816V-positive    mastocytosis: waiting for proof-of-principle. Leukemia and Lymphoma.    March 2010; 51(3): 360-362.-   Miettinen et al. KIT (CD117): A Review on Expression in Normal and    Neoplastic Tissues, and Mutations and their Clinicopathologic    Correlation. Appl Immunohistochem Mol Morphol. 2005; 13: 205-220.-   Hug et al. ETO Interacting Proteins. Oncogene. 2004; 23(24):    4270-4274.-   Akin et al. A Novel Form of Mastocytosis Associated with a    Transmembrane C-KIT Mutation and Response to Imatinib. Blood. 2004;    103: 3222-3225.-   Zermati et al. Effect of Tyrosine Kinase Inhibitor STI571 on the    Kinase Activity of Wild-type and Various Mutated C-KIT Receptors    Found in Mast Cell Neoplasms. Oncogene. 2003; 22: 660-664.-   Akin et al. Effects of Tyrosine Kinase Inhibitor STI571 on Human    Mast Cells Bearing Wild-type or Mutated C-KIT. Exp Hematol. 2003;    31: 686-692.-   Ma et al. The C-KIT Mutation Causing Human Mastocytosis is Resistant    to STI571 and Other KIT Kinase Inhibitors; Kinases with Enzymatic    Site Mutations Show Different Inhibitor Sensitivity Profiles than    Wild-type Kinases and Those with Regulatory-type Mutations. Blood.    2002; 99: 1741-1744.-   Davis M I, Hunt J P, Herrgard S, et al. Comprehensive analysis of    kinase inhibitor selectivity. Nat Biotechnol 2011; 29:1046-51.

What is claimed is:
 1. A method of inhibiting or reducing mutant C-KITtyrosine kinase activity or expression in a subject suffering from aproliferative disease comprise of identifying the subject having aproliferative disease positive for a constitutively active mutant C-KITor over expression of C-KIT comprising administering to the subjecthaving or suspected to have the proliferative disease, a therapeuticallyeffective amount of the compound of Formula I:

or a pharmaceutically acceptable salt or solvate thereof, wherein theC-KIT mutant is located in at least one of exon 8, exon 9, exon 11, exon13, exon 14, or exon
 17. 2. The method of claim 1, wherein the C-KITmutant is at least one of T417I, Del 418, Del 418-419, T417I, T417V,T417R, T417N, Y418R, Y418A, Y418G, R420W, W420G, L421G, L421F, D496V,Ins A504_Y505, Del 557-558, V559A, V559D, K642E, V560G, V654A, T670I,D816E, D816I, D816V, D820E, D820Y, D820V, N822H, N822K, Y823C, Y823D,A829P, V559D/T670I, V559D/V654A, V560G/D816V, and V560G/N822K mutantC-KIT.
 3. The method of claim 1, wherein the compound is provided forthe treatment of secondary mutations, wherein secondary mutations is atleast one of T670I, V654A, D816V, and N822K.
 4. The method of claim 1,wherein the proliferative disease is selected from at least one ofmastocytosis, acute myeloid leukemia, gastrointestinal stromal tumors,sinonasal NK/T-cell lymphoma, seminomas, dysreminomas, melanomas, andthymic carcinomas.
 5. The method of claim 1, wherein the therapeuticallyeffective amounts are from about 15 to 500, 25 to 450, 50 to 400, 100 to350, 150 to 300, 200 to 250, 15, 25, 50, 75, 100, 150, 200, 250, 300,400, 450, or 500 mg per day.
 6. The method of claim 1, wherein thecompound is administered at least one of continuously, intermittently,systemically, or locally.
 7. The method of claim 1, wherein the mutantC-KIT is defined further as constitutively active.
 8. The method ofclaim 1, wherein the compound is administered orally, intravenously, orintraperitoneally.
 9. The method of claim 1, wherein the Crenolanib isCrenolanib Besylate, Crenolanib Phosphate, Crenolanib Lactate,Crenolanib Hydrochloride, Crenolanib Citrate, Crenolanib Acetate,Crenolanib Toluenesulphonate or Crenolanib Succinate.
 10. The method ofclaim 1, wherein the therapeutically effective amount of compound isadministered up to three times or more a day for as long as the subjectis in need of treatment for a proliferative disease.
 11. The method ofclaim 1, wherein the compound is provided at least one of sequentiallyor concomitantly, with another pharmaceutical agent in a newly diagnosedproliferative disease subject, to maintain remission, or arelapsed/refractory proliferative disease subject.
 12. The method ofclaim 1, wherein the compound is provided as a single agent or incombination with another pharmaceutical agent in a newly diagnosedproliferative disease subject, to maintain remission, or arelapsed/refractory proliferative disease subject.
 13. The method ofclaim 1, wherein the compound is provided as a single agent or incombination with another pharmaceutical agent in a newly diagnosedproliferative disease pediatric subject, to maintain remission, or arelapsed/refractory proliferative disease pediatric subject.
 14. Themethod of claim 1, further comprising the step of determining if thesubject is relapsed/refractory to interferon alpha,2-chlorodoxyadenosine or Imatinib mesylate prior to providing thecompound.
 15. A method for treating a subject suffering from a C-KITmutated proliferative disease comprising: administering to the subjectin need of such treatment a therapeutically effective amount ofCrenolanib or a salt thereof, wherein the cell proliferative disorder ischaracterized by mutant or double mutant C-KIT receptor tyrosine kinaseactivity, proliferative disease is selected from at least one of amastocytosis, acute myeloid leukemia, gastrointestinal stromal tumors,sinonasal NK/T-cell lymphoma, seminomas, dysgerminomas, melanomas, andthymic carcinomas. wherein the mutant or double mutant is at least oneof: mutations in exons 8, 9, 11, 13, 14, or 17, T417I, Del 418, Del418-419, T417I, T417V, T417R, T417N, Y418R, Y418A, Y418G, R420W, W420G,L421G, L421F, D496V, Ins A504_Y505 Del 557-558, V559A, V559D, K642E,V560G, V654A, T670I, D816E, D816I, D816V, D820E, D820Y, D820V, N822H,N822K, Y823C, Y823D, A829P, V559D/T670I, V559D/V654A, V560G/D816V, andV560G/N822K mutant C-KIT.
 16. The method of claim 15, wherein thecompound is provided for the treatment of secondary mutations from atleast one of the following mutations T670I, V654A, D816V, and N822K. 17.The method of claim 15, wherein the compound is administered orally,intravenously, or intraperitoneally.
 18. The method of claim 15, whereinthe Crenolanib is Crenolanib Besylate, Crenolanib Phosphate, CrenolanibLactate, Crenolanib Hydrochloride, Crenolanib Citrate, CrenolanibAcetate, Crenolanib Toluenesulphonate or Crenolanib Succinate.
 19. Themethod of claim 15, wherein the therapeutically effective amount of thecompound is administered up to three times or more a day for as long asthe subject is in need of treatment for proliferative disease.
 20. Themethod of claim 15, wherein the Crenolanib is provided at least one ofsequentially or concomitantly, with another pharmaceutical agent in anewly diagnosed C-KIT mutated proliferative disease, to maintainremission, or a relapsed/refractory C-KIT mutated proliferative disease.21. The method of claim 15, wherein the Crenolanib is provided as asingle agent or in combination with another pharmaceutical agent fortreatment of a pediatric subject with the proliferative disease.
 22. Themethod of claim 15, wherein the Crenolanib is provided as a single agentto at least one of post chemotherapeutic or targeted therapy in newlydiagnosed C-KIT mutated proliferative disease.
 23. The method of claim15, wherein the Crenolanib is provided as a single agent in treatment ofsubjects with the C-KIT mutated proliferative disease that is eitherrefractory to or has relapsed after treatment with anotherchemotherapeutic or targeted therapy.
 24. The method of claim 15,wherein the subject is refractory to at least one of interferon alpha, 2chlorodoxyadenosine or Imatinib mesylate.
 25. The method of claim 15,further comprising the step of determining identifying a subject in needof cancer therapy caused by uncontrolled C-KIT tyrosine kinase activityor expression.